| BackgroundDiffuse large B cell lymphoma (DLBCL) is the most common Hodgkin's disease, which accounts for 30%～40% of adult lymphoma.DLBCL is a group of heterogeneous disease, whether cell sources, clinical manifestation, reaction to chemotherapy or prognosis, and also there are different between individual. DLBCL clinical manifestation show certain aggressive, although most DLBCL patients react to the traditional combination chemotherapy such as CHOP, but the really cure patients are less than half. Relapse and refractory on DLBCL patients is true a huge challenge for us. Application of Rituximib had greatly improved the therapeutic effect in patients with DLBCL, especially refractory or relapse DLBCL. By cDNA microarray and oligonucleotide microarray technology, DLBCL divided from molecular biology admiral born three subtypes:germinal center B cells lymphoma (GCB-DLBCL) and activation B cell lymphoma (ABC-DLBCL)-and the three type. In 2004 Hans et al detected three antibodies CD10,Mum-land BCL-6 by immunohistochemistry and divided DLBCL into two major subtypes GCB type(CD10+BCL-6+ or CD10-BCL-6+MUM1-) and non-GCB type (CD10-BCL-6-or CD10-BCL-6+MUM1+). GCB type and non-GCB type is original from normal germinal B cell and activated peripheral B cell respectively, and they have different gene expression profiling. As for the prognostic, they have different progress. Cell adhesion molecules are a transmembrane glycoprotein which are closely related to tumor invasive, metastasis. CD44 protein is a more common form of cell adhesion proteins, which are widely distributed in the cell surface, like other adhesion molecules, which are primarily involved in cell-cell, cell matrix with the outside environment, adhesion and signal transduction. Generally,the standard CD44 molecule contain three part:extracellular domain, transmembrane domain, intracellular domain (CD44ICD). Extracellular domain of CD44 contains hyaluronic acid binding site, which is the major components of the extracellular matrix, through combination with hyaluronic acid cells can adhesion around the matrix. With the help of relevant protease, CD44 through a series orderly hydrolysis, and gradual release the extracellular structures and the CD44ICD. The role of CD44 extracellular domain and CD44ICD are different. After extracellular release the soluble CD44 fragment (sCD44) formation. sCD44 can bind hyaluronic acid even after the release, and by competitive binding hyaluronic acid with CD44, it play as an interfering role of cell adhesion. Under the action of the y-secretase enzyme CD44ICD is released from the cell membrane and positioning in the nucleus, and participate in transcription regulation, which including CD44 itself.Cell signal transcription and activate protein family (STATs) is a group proteins in cells related to transcriptional. Many cell stimulating factor and growth factors through the cell surface receptors can stimulate STATs way. Stat3 the most easily occurred a member phosphorylation in the family of STATs which is exists in cell plasma and related to the tyrosine phosphorylation signal paths. Stat3 expression can be induced to increase or continuous activation cell proliferation, prevent cell apoptosis, increase cell transformation ability. By inhibiting Stat3 express or reduce the activated Stat3 level is one of the new direction of cancer treatment. Stat3 apart as transcription factors in the cell plasma, also undertakes the important role, this two joint role directly lead to cancer occurs, the invasion and metastasis.Nuclear transcription factors-κB (NF-κB) is widely exists in eukaryotic cells and it is a induced transcription factor family protein. In most cells, NF-κB combined with its inhibiting protein familyκB IkBs located in cell plasma.When NF-κB occur phosphorylation, IkBs drop solution, NFκB-NF-κB from cell plasma transship to the nucleus, and bind with the DNA sequence in the nucleus, and up-regulation the target genes expression, however the down-regulate and feedback adjustment effect also can make NF-κB to restrain state. NF-κB's two-way regulating functions is broken in most tumor cells, and is continuous activated. This effect may inhibit some gene expression, which basically participate in the cell cycle signal, apoptosis, and cell adhesion and cell migration. Thus NF-κB is closely related to the occurrence and development of tumor.ObjectiveThe progression and chemoresistance of DLBCL, partly correlated with the abnormal of transcription in tumor cell.which reveal that regulation the transcription is one of the ways to cure relapse/refractory tumor such as DLBCL.Our study want to kown whether CD44ICD exist in DLBCL cells and then observe the CD44ICD location. Step to investigate CD44ICD function in ABC-DLBCL migrate, grow, uncovered the relation among the NF-κB, Stat3 and explore the mechanismMaterials and Method1. Cell lines:ABC-DLBCLcells:SUDLH2,LY3;GCB-DLBCL cells:LY8.2. Flow cytometric analysis the surface expression of CD44 in SUDLH2,Ly3,Ly8 cell lines.3. Western blotting detected the expression and location of CD44ICD in SUDLH2 cells, know inhibit dose of DAPT for CD44ICD.4. Western bloting analysis of CD44ICD, NF-κB, Stat3 protein expression in SUDLH2,LY8 cells after 10 u M DAPT treatment or no.5. MTT analysis of cytotoxicity of serial concentrations of DAPT to SUDLH2 and LY8 cells at 24h,48h, and 72h.6. Transwell to detect the migration of SUDLH2,LY8 cells after 0μM,10μM,100μM DAPT treatment or no.7. The statistical analyses were performed with the statistical software package SPSS 13.0. One-Way ANOVA was used to compare the difference of Transwell population and Univariate used to apoptotic population of DLBCL cells and Bonferroni was used to do multiple comparison when the variance was homogenous,if not,Dunnett's T3 was employed.A value of *P<0.05 was accepted as an indication of statistical significance. Results represent the mean±SD of at least three independent experiments.Result1. Flow cytometric analysis detectin the level of CD44 expression on three DLBCL cells surface Flow cytometry analysis show that the expression of CD44 in SUDHL2, Ly3, but not in Ly8 cells. The expression of CD44 on the three cells were (74.57±5.03)%, (44.01±3.70)%, (0.14±0.03)%, individual.2. The expression and positioning of CD44ICD in SUDLH2 Western Bloting detects CD44ICD in both plasma and nucleus in SUDLH2 cells and mainly located in the nucleus and 5 MμDAPT can inhibit the produce of CD44ICD in SUDLH2 cells.3. The signaling protein expression in two subtypes DLBCL cells after inhibit the CD44ICD expression with DAPT 10μM DAPT treatment inhibit the expression of CD44ICD, Stat3, NF-κB in SUDLH2 and Ly3 cells.CD44ICD, Stat3 was not detected in Ly8 cell. 10μM DAPT was without obvious influence the NF-κB expression in Ly8 cells.4. The cells proliferation of two subtypes DLBCL after inhibit the CD44ICD expression with DAPT 0.1 u M,1μM,10μM, DAPT without significant inhibitory effect on two kinds of cells,100 M DAPT have significant inhibition on two cell lines, after 48 hours both cell lines have obvious inhibition.5. The alter of cell migration after inhibit the CD44ICD expression with DAPT The effection of DAPT on ABC- DLBCL cells' migration Inhibit CD44ICD expression on ABC-DLBCL cells, as compared with control subjects the migration was no change after 12 hours with 10μM,100μM DAPT treatment,both in GCB-DLBCL or ABC-DLBCL cell.Conclusion1,CD44ICD was detected in ABB-DLBCL cells SUDLH2;2,CD44ICD was detected both in plasma and nucleu of SUDLH2 cells,but mainly located in nucleus positioning in;3,γsecretase inhibitors DAPT can inhibitor the CD44ICD expression in SUDLH2 cells, obviously;4,CD44ICD may participate in adjusting the expression of Stat3, NF-κB in ABC-DLBCL cells;5,CD44ICD DLBCL was without obvious effection in proliferation and migration function in ABC -DLBCL cells. |
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